Gene encoding HM1.24 antigen protein and promoter thereof

ABSTRACT

There are provided a genomic DNA comprising 4 exons encoding the amino acid sequence as set forth in SEQ ID NO: 2 and 3 introns ligating them, and a splicing variant of said genomic DNA; as well as a DNA having the base sequence as set forth in SEQ ID NO: 4 and a promoter activity and the fragment thereof, and uses thereof.

TECHNICAL FIELD

The present invention relates to a genomic gene encoding HM1.24 antigenprotein, a promoter of the gene encoding HM1.24 antigen protein, anduses thereof.

BACKGROUND ART

Mouse anti-HM1.24 monoclonal antibody has been prepared using a humanmyeloma cell line KPC-32 as an immunogen (Goto, T. et al., Blood 84:1922-1930, 1994). The HM1.24 antigen that is recognized by this antibodyis a membrane protein having a molecular weight of 29 to 33 kDa that isoverexpressed on the surface of myeloma cells. Furthermore, for normalcells its expression has been confirmed in immunoglobulin-producing Bcells (plasma cells, lymphoplasmacitoide cells), but expression israrely observed in the other cells and tissues (Goto T. et al., supra).However, nothing is known about HM1.24 antigen except its expressiondistribution and molecular weight.

According to the present invention, as a result of the cloning ofgenomic DNA encoding HM1.24 antigen, the determination of its nucleotidesequence and the deduced amino acid sequence, and further homologysearch, HM1.24 antigen was demonstrated to be a molecule identical withBST2 that is a surface antigen expressed on the stroma cells isolatedfrom the bone marrow of patients with myeloma, and the bone marrow andthe snynovial membrane of patients with rheumatoid arthritis. BST2 hasbeen shown to have an ability of supporting the growth of pre-B cellsand is thought to be involved in the pathology of rheumatoid arthritis,but its other physiological functions are not known (Ishikawa J. et al.,Genomics 26: 527-534, 1995).

In the production of a useful gene product derived from an animal bymeans of genetic engineering, it often happens that the gene is notexpressed, a gene product, protein, does not take a correctconformation, post-translational modification does not occur correctlyand the like, when a microorganism host such as Escherichia coli,Bacillus subtilis, or yeast is used. In order to solve such problems,animal cells are often used as hosts, in which case, the selection of apromoter has a great impact on expression efficiency. Conventional,frequently used promoters for animal cells include SV40 promoter,cytomegalovirus promoter, actin promoter, and the like.

DISCLOSURE OF THE INVENTION

Considering the above state of art, the present invention provides agenomic DNA encoding HM1.24 antigen protein.

The present invention also provides a process for producing HM1.24antigen protein using animal cells by means of said genomic DNA.

When a useful gene product is to be produced in large quantities usinganimal cells as a host, conventionally used promoters for animal cellsare not always satisfactory in terms of transcription activity, andhence there is a great need for the development of stronger promoters.Thus, it is an object of the present invention to provide a DNA having astronger promoter activity as a promoter for animal cells and usesthereof.

In order to solve the above problems, the present invention provides agenomic DNA encoding HM1.24 antigen protein, said DNA comprising 4 exonregions encoding the amino acid sequence as set forth in SEQ ID NO: 2.As an example of the above genomic DNA, the present invention provides agenomic DNA having 4 exons encoding the amino acid sequence and 3introns as set forth in SEQ ID NO: 2.

The present invention also provides a splicing variant of the abovegenomic DNA. Specific examples include a splicing variant lacking exon2, a splicing variant lacking exons 2 and 3, and the like.

The present invention also provides a process for producing HM1.24antigen protein which method comprises culturing animal cellstransformed with an expression vector comprising the above genomic DNA.

The present invention further provides a promoter sequence DNA havingthe nucleotide sequence of the 5′-non-coding region as set forth in SEQID NO: 4 or a DNA fragment of said sequence having a promoter activityin animal cells.

The present invention also provides a DNA that hybridizes with the aboveDNA or a fragment thereof under a stringent condition and that has apromoter activity in animal cells. The above DNA having promoteractivity is preferably derived from animal cells, in particularmammalian cells.

The present invention also provides a DNA that has been modified by thedeletion, addition and/or substitution with other nucleotides, of one ora plurality of nucleotides in the nucleotide sequence of the5′-non-coding region as set forth in SEQ ID NO: 4 and that has apromoter activity in animal cells.

The present invention also provides a recombinant DNA wherein a usefulgene is operably linked to the above DNA having a promoter activity. Asthe above useful genes, there can be mentioned, for example, nucleicacids selected from the group consisting of nucleic acids encodinguseful proteins, antisense DNA, antisense RNA, nucleic acids encodingdecoys, and ribozyme.

The present invention also provides a vector comprising the aboverecombinant DNA. The vector is a plasmid vector or a virus vector.

The present invention also provides animal cells into which the aboverecombinant DNA has been introduced.

The present invention also provides animal cells that have beentransformed with the above vector.

The present invention also provides an animal having the above animalcells.

The present invention also provides a method of expressing a useful genewhich method comprises culturing animal cells into which the aboverecombinant DNA has been introduced.

The present invention also a process for producing a useful proteinwhich process comprises culturing animal cells transformed with anexpression vector comprising a nucleic acid encoding a useful proteinoperably linked to the above DNA having a promoter activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing that shows the nucleotide sequence of cDNA (SEQ IDNO: 1) encoding HM1.24 antigen protein and the corresponding amino acidsequence (SEQ ID NO: 3). The underlined part shows a N-type sugar chainbinding motif.

FIG. 2 is a drawing that shows the nucleotide sequence of Cdna (SEQ IDNO: 1) encoding HM1.24 antigen protein and the corresponding amino acidsequence (SEQ ID NO: 3).

FIG. 3 is a schematic diagram showing a clone P3.19 isolated using thepanning method and 5 clones (IS1 to IS5) isolated by the immunoscreeningmethod.

FIG. 4 is a drawing that shows the result of flow cytometry analysisusing anti-HM1.24 antibody (A: CHO/NEO, B: CHO/HM). The histogram ofanti-HM1.24 antibody is shown by a solid line, and that of the controlantibody (UPC10) that showed a matching isotype is shown by a brokenline. In the figure, the abscissa refers to fluorescence intensity andthe ordinate to cell count.

FIG. 5 is a photograph in which the expression of HM1.24 antigen in eachcell line and HM1.24 antigen-expressing CHO cells was detected by theimmunoprecipitation/Western blotting method using anti-HM1.24 antibody.After immunoprecipitation using the anti-HM1.24 antibody-bound Sepharose4B (lanes 1-6) or unbound Sepharose 4B (lanes 7 and 8), Western blottingwas carried out using anti-HM1.24 antibody to detect HM1.24 antigen(shown on the right). (*: anti-HM1.24 antibody H chain).

FIG. 6 is a drawing that shows a restriction map of the 5′-untranslatedregion comprising the promoter region of the HM1.24 antigen proteingene.

FIG. 7 is a drawing that shows the nucleotide sequence (SEQ ID NO: 4) ofthe 5′-untranslated region comprising the promoter region of the HM1.24antigen protein gene. Each transcription factor binding motif has beenunderlined.

FIG. 8 is a drawing that shows the nucleotide sequence (SEQ ID NO: 4) ofthe 5′-untranslated region comprising the promoter region of the HM1.24antigen protein gene. Each transcription factor binding motif has beenunderlined, the TATA-like sequence has been boxed, the transcriptioninitiation point is represented by an arrow, and the region encoding 7amino acids at the N-terminal of the protein is represented by theone-letter code of amino acids (SEQ ID NO: 21).

In FIG. 9, (A) shows the position of a primer corresponding to thegenonie encoding HM1.24 antigen protein, and (B) shows the base sequence(SEQ ID NOs: 9-16) of each primer.

FIG. 10 is a drawing that shows a restriction map of genomic DNAencoding HM1.24 antigen protein and the positions of the correspondingexons and introns.

FIG. 11 is a drawing that shows a restriction map of genomic DNA (SEQ IDNO: 2) encoding HM1.24 antigen protein and the corresponding amino acidsequence (SEQ ID NO: 3) (upstream side). The arrow shows thetranscription initiation point and the underline shows the N-type sugarchain binding motif.

FIG. 12 is a drawing that shows the nucleotide sequence of genomic DNA(SEQ ID NO: 2) encoding HM1.24 antigen protein and the correspondingamino acid sequence (SEQ ID NO: 3) (downstream side). The doubleunderline shows the poly A-addition signal.

FIG. 13 is a drawing that shows the nucleotide sequence (SEQ ID NO: 19)of a splicing variant of human HM1.24 antigen protein and thecorresponding amino acid sequence (SEQ ID NO: 20). The underlined partshows where the amino acid sequence is different from that of humanHM1.24 antigen protein.

FIG. 14 is a drawing that shows the nucleotide sequence (SEQ ID NO: 33)of the genomic DNA of HM1.24 antigen protein. A genome was present whichhas mutations, a.fwdarw.g at position 178, g.fwdarw.a at position 262,and t.fwdarw.c at position 323, as well as a deletion of one of 9 a'snear position 360. The symbol “*” represents a transcription initiationpoint. There was also a genome in which 19 bp at positions 93-111 repeatin tandem. The symbol “.fwdarw.” shows the position of the sense primer.

FIG. 15 shows the nucleotide sequence of genomic DNA of HM1.24 antigenprotein and the corresponding amino acid sequence. There was also agenome in which 8 base pairs at positions 551-558 were deleted. Thesymbol “←” shows the position of the antisense primer.

FIG. 16 is a drawing that shows the nucleotide sequence (SEQ ID NO: 33)of genomic DNA (intron site) of HM1.24 antigen protein. The .fwdarw.shows the position of the sense primer.

FIG. 17 shows the nucleotide sequence of genomic DNA of HM1.24 antigenprotein and the corresponding amino acid sequence. The → shows the senseprimer, and the ← indicates the antisense primer.

FIG. 18 shows the nucleotide sequence of genomic DNA of HM1.24 antigenprotein and the corresponding amino acid sequence. There was also agenome in which 3 out of 5 c's near position 2315 were deleted. The ←shows the position of the antisense primer.

EMBODIMENT FOR CARRYING OUT THE INVENTION

A genomic gene comprising a genomic DNA and a promoter region of humanHM1.24 antigen can be easily amplified by a PCR method using suitableprimers. Thus, a genomic DNA of human HM1.24 antigen can be amplified bydesigning a sense primer that hybridizes to the 5′-end of a genomic DNAsequence as set forth in SEQ ID NO: 2 and an antisense primer thathybridizes to the 3′-end, and then by performing a PCR reaction using apolymerase, such as AmpliTaq (Perkin Elmer), LA-Taq (Takara Shuzo), andthe like, and using as a template human genomic DNA prepared accordingto a standard method. A PCR product can be directly inserted into acloning vector such as pCRII (Invitrogen) or pGEM-T (Promega).

By introducing a restriction enzyme recognition site into a sense primeror an antisense primer, it can be inserted into a desired vector.

Genomic DNA that contains a promoter region of human HM1.24 antigen canalso be amplified by the same method. Thus, a desired DNA fragment canbe obtained by designing a sense primer that hybridizes to the 5′-end ofthe sequence as set forth in SEQ ID NO: 4 and an antisense primer thathybridizes to the 3′-end, and then by amplifying by a PCR reaction usinghuman genomic DNA as a template.

A genomic DNA encoding HM1.24 antigen protein of the present inventioncomprises, as shown in FIG. 10, 4 exons and 3 introns linking them, andtheir specific nucleotide sequences and deduced amino acid sequences ofthe exon regions are as shown in FIGS. 11 and 12 (SEQ ID NO: 2). Thus,exon 1 encodes from amino acid Met at position 1 to amino acid Val atposition 95; exon 2 encodes from amino acid Met at position 96 to aminoacid Glu at position 117; exon 3 encodes from amino acid Glu at position118 to amino acid Arg at position 138; and exon 4 encodes from aminoacid Arg at position 139 to amino acid Gln at position 180.

The present invention also provides splicing variants of genomic DNAencoding HM1.24 antigen protein. Splicing variants are those in which atleast one, i.e. 1 to 3, of exons 1 to 4 has been removed, for exampleexon 2 or 3, or both of them are removed.

The present invention also provides splicing variants of genomic DNAencoding HM1.24 antigen protein having a nucleotide sequence of DNA inwhich the codon corresponding to each amino acid of at exon is out ofposition because the nucleotide sequence in an exon was deleted due tosplicing.

Since the splicing variants have the reading frames of different aminoacid sequences, they have amino acid sequences different from that ofHM1.24 antigen protein encoded by exons 1 to 4. As an example of suchsplicing variants, there can be mentioned a splicing variant having thenucleotide sequence and the amino acid sequence as set forth in SEQ IDNO: 17.

A genomic DNA encoding the HM1.24 antigen protein of the presentinvention can be obtained by cloning a cDNA that encodes HM1.24 antigenprotein, then using this cDNA to design a primer oligonucleotide, whichis amplified by the PCR method using genomic DNA as a template. In orderto clone cDNA, animal cells expressing HM1.24 antigen, for example KPMM2cells, are cultured, and from the cell culture total RNA is extractedaccording to a standard method and then mRNA is enriched.

According to the present invention, based on the above mRNA, cDNA issynthesized by a standard method, which is fractionated using alow-melting point agarose gel. Then cDNA having a size of 0.7 kbp orgreater is inserted into an expression vector pCOS1 or λExCell vector toprepare a library A which is used for screening by direct expressioncloning, i.e. panning, and library B which is used for immunoscreening.

For screening the panning method an expression plasmid that constituteslibrary A was in introduced into COS-7 cells using electroporation.After culturing, attached cells were scraped off and were contacted to apanning plate coated with anti-HM1.24 antibody to allow the cellsexpressing HM1.24 to be attached to the plate. Then plasmid DNA wasextracted from the cells attached to the plate, amplified in E. coli,and used for the subsequent panning. The panning procedure was repeatedthree times to select clones that express antigen reacting withanti-HM1.24 antibody, and one of the clones was designated as cloneP3.19.

Sequencing revealed that clone P3.19 consists of 1,012 bp and containsan open reading frame encoding 180 amino acids. The nucleotide sequenceof the cDNA insert in this clone P3.19 and the corresponding amino acidsequence are shown in FIG. 1 and SEQ ID NO: 1. The amino acid sequencealone is shown in SEQ ID NO: 3.

For immunoscreening, on the other hand, a phage constituting library Bwas cultured together with E. coli NM522 on an agar plate, theexpression product was transferred to a nitrocellulose filter, and thefilter was contacted to an anti-HM1.24 antibody solution. Anti-HM1.24antibody that was bound to the filter via binding with the expressionproduct was detected with labeled anti-mouse immunoglobulin (Ig) serum.

This produced 5 positive clones: IS-1 to IS-5. The nucleotide sequencesof these cDNA inserts-were determined and were compared to thenucleotide sequence of the cDNA of the above P3.19. The comparisonrevealed, as shown in FIG. 3, that any cDNA in clones IS-1 to IS-5 waspart of the cDNA of P3.19 and the 5′-end has been deleted in P3.19.

Then, after P3.19 was introduced into CHO cells to transform the cells,flow cytometry was performed using anti-HM1.24 antibody. The resultconfirmed, as shown in FIG. 4, that HM1.24 antigen was expressed.Furthermore, as shown in FIG. 4, P3.19 was confirmed to encode HM1.24antigen by immunoprecipitation as well.

Then, as shown in FIG. 9A, the cDNA sequence was divided into fourregions, and primer pairs were designed as shown in FIG. 9B to amplifyeach part. The genome library prepared according to a standard methodwas PCR-amplified using the above each pair of primers, which were thenligated together to obtain a full-length genomic DNA.

The result is shown in FIGS. 11 and 12, and SEQ ID NO: 2. As can beseen, the genomic DNA encoding HM1.24 antigen protein has 4 exons and 3introns linking them. These relationships are schematically shown inFIG. 10, which also shows a restriction map of genomic DNA.

The present invention also relates to a process for producing HM1.24antigen protein which method comprises culturing animal cellstransformed with an expression vector into which the above genomic DNAhas been inserted. As animal cells for use in this process, variousanimal cells, for example, described below with respect to the promotersof the present invention may be used, and cell cultures of humans,mammals other than humans, insects and the like are preferred. Forexample, HeLa etc. are used as the cell culture of humans; CHO, COS,myeloma, BHK, Vero, etc. are mentioned as the cell cultures of mammalsother than humans; and cell cultures of silkworm etc. are mentioned asthe cell cultures of insects. As vectors for introducing DNA encodingthe HM1.24 antigen protein of the present invention into animal cells,for example phage vectors such as M13 are used.

Culturing of animal cells for producing HM1.24 antigen protein can beperformed according to a standard method, and the isolation of HM1.24antigen protein from the culture can also be performed according to astandard method.

Hybridoma HM1.24 producing mouse anti-HM1.24 monoclonal antibody thatspecifically recognizes HM1.24 antigen protein has been internationallydeposited under the provisions of the Budapest Treaty as FERM BP-5233 onSep. 14, 1995 with the National Institute of Bioscience and HumanTechnology, Agency of Industrial Science and Technology, of 1-3, Higashi1-chome, Tsukuba-shi, Ibaraki pref., Japan.

The promoter of the present invention and the uses thereof will now beexplained below.

The word “promoter” as used herein includes, but is not limited to, aregion that is located 20-30 base pairs upstream to the transcriptioninitiation point (+1) and that includes a TATA box or a TATA box-likeregion responsible for directing RNA polymerase to start transcriptionat a correct position, and, in addition to this region, it may includeregions that are required for proteins, other than RNA polymerase, toassociate with for adjusting expression. When the term “promoter region”is used in the present invention, it means a region that includes thepromoter as used herein.

The words “promoter activity” as used herein means an ability or afunction of being ligated to a useful gene downstream to the promoter ina state that enables expression, so that when introduced into a host(animal cell) it can produce either intracellularly or extracellularly agene product of the useful gene. In general, the presence or absenceand/or intensity of a promoter is expressed as the promoter activity byligating, downstream to the promoter, a gene (reporter gene) encoding aneasily quantifiable protein in a state that enables expression,introducing it into a host, and then determining the amount expressed ofthe protein. Thus, when the expression of gene products of a useful genewere confirmed either intracellularly or extracellularly after theuseful gene was ligated downstream to the promoter and introduced into ahost, the promoter should have a promoter activity in the host intowhich the gene was introduced.

The words “animal cells” as used herein includes cells derived fromhumans, but they are not limited to them as long as the promoter of thepresent invention has a promoter activity in an animal cell. Forexample, there can be mentioned mammals other than humans (for example,mice, rats, rabbits, goat, pigs, cattle, horses, dogs, monkeys, andchimpanzees), birds (for example, chickens, turkeys, quails, ducks, andgeese), reptiles (for example, snakes, crocodiles, and turtles),amphibians (for example, frogs, salamanders, and newts), fish (forexample, scads, mackerel, sea bass, sea breams, sea perch, yellowtails,tuna, salmon, trout, carp, sweetfish, eel, soles, sharks, rays, andsturgeons).

The words “useful genes” as used herein includes, for example, nucleicacids encoding proteins that can be expressed in animal cells, antisenseDNA or sense DNA of genes derived from animal cells, nucleic acidsencoding decoys that have genes encoding the binding proteins oftranscription factors derived from animal cells or sequences of thebinding sites of transcription factors or similar sequences, andribozymes that cleave mRNA derived from animal cells.

As nucleic acids encoding protein that can be expressed in animal cellsinclude, but not limited to, those derived from animals, and as long asthey can be expressed in animal cells, those derived from microorganismssuch as bacteria, yeasts, actinomycetes, fungi, Ascomycetes, andBasidiomycetes, or those derived from living organisms such as plantsand insects are also included in the useful genes mentioned in thisspecification.

The words “nucleic acids encoding decoys” as used herein means DNA thathave genes encoding the binding proteins of transcription factorsderived from animal cells or sequences of the binding sites oftranscription factors or similar sequences, which are introduced as“decoys” into cells so as to suppress the action of the transcriptionfactors. The word “ribozymes” as used herein means those that cleavemRNA of specific proteins, and that inhibits the translation of thesespecific proteins.

Ribozyme can be designed from gene sequences encoding specific proteinsand, for a hammerhead type ribozyme, for example, the method asdescribed in FEBS Letter, 228: 228-230 (1988) can be used. In additionto the hammerhead type ribozymes, any type of ribozymes including thehairpin type ribozymes, the delta type ribozymes, and the like thatcleave mRNA of these specific proteins and that inhibit the translationof these specific proteins can be included in the ribozyme as usedherein.

By ligating a useful gene downstream to a DNA fragment having thepromoter activity of the present invention in a state that enablesexpression, the expression of the useful gene can be enhanced. Thus,useful genes are expressed in animal cells into which recombinant DNAcomprising DNA having the promoter activity of the present invention anda useful gene ligated in a state that enables expression has beenintroduced with or without using a vector. As a vector, a plasmidvectors or a virus vectors is preferably used. When vectors are notused, DNA fragments can be introduced according to the methods describedin the literature [Virology, 52: 456 (1973); Molecular and CellularBiology, 7: 2745 (1987); Journal of the National Cancer Institute, 41:351 (1968); EMBO Journal, 1: 841 (1982)].

Animal cells having such recombinant DNA fragments of the presentinvention and animals having such animal cells are also encompassed inthe scope of the present invention. As useful genes whose expression canbe enhanced according to the present invention, there can be mentioned,as described above, DNA encoding protein, antisense DNA, antisense RNA,polynucleotides encoding a decoy, nucleotide sequences functioning as adecoy, ribozymes, and the like. The present invention also disclosesmethods of producing proteins of interest, and methods of expressinguseful genes using a DNA fragment having the promoter activity of thepresent invention.

Thus, a process for producing protein is also encompassed in the scopeof the present invention wherein said process comprises ligating anucleic acid encoding protein downstream to DNA having the promoteractivity of the present invention in a state that enables expression,culturing an animal cell transformed with a vector containing therecombinant DNA thus obtained, and harvesting said protein from theculture. Similarly, a method of expressing a useful gene comprisingligating a useful gene downstream to DNA having the promoter activity ofthe present invention in a state that enables expression, introducingthe recombinant DNA thus obtained into an animal cell, and culturing, orthe method of expressing a useful gene using an animal cell said methodcomprising transforming an animal cell with a vector containing saidrecombinant DNA and culturing said animal cell is also encompassed inthe scope of the present invention.

DNA having the promoter activity of the present invention is DNA havingthe nucleotide sequence shown in the 5′-end non-coding region as setforth in SEQ ID NO: 4 or a fragment thereof retaining a promoteractivity. The 5′-end non-coding region means the nucleotide sequence upto position 2040 in SEQ ID NO: 4. It is known that a size of 5nucleotides or greater is required to exhibit a promoter activity inanimal cells. Thus, fragments of DNA having the promoter activity of thepresent invention have a size of at least 5 nucleotides or greater,preferably 30 nucleotides or greater, and more preferably 2000nucleotides or greater.

The present invention also includes DNA that can hybridize with DNAhaving the nucleotide sequence as set forth in SEQ ID NO: 4 under astringent condition and that has a promoter activity. The hybridizingDNA is for example a genomic DNA library derived from natural sources,for example mammals such as humans, mice, rats, and monkeys. As astringent condition, there may be mentioned for example a low stringentcondition. By way of example, a low stringent condition is a washingcondition provided by 42° C. in 5×SSC, 0.1% sodium dodecyl sulfate, and50% formamide. More preferably, a high stringent condition may bementioned. By way of example, a high stringent condition is a washingcondition provided by 60° C. in 0.1×SSC and 0.1% sodium dodecyl sulfate.

The present invention also includes a DNA fragment that has beenmodified by the deletion, addition, and/or substitution with otherbases, of one or a plurality of nucleotides in the nucleotide sequenceof the promoter as set forth in SEQ ID NO: 4 and that retains a promoteractivity. The degree of modification is in the range of 70% homology tothe nucleotide sequence as set forth in SEQ ID NO: 4, preferably ahomology of 80% or greater, and more preferably a homology of 90% orgreater.

“Homology” as used herein means the degree of identity of residuesexhibited by two or more non-complementary nucleotide sequences or aminoacid sequences (Gene Cloning 2nd edition, T. A. Brown, Chapman and Hall,1990). Thus, a homology of 90% means that 90 residues or more out of 100are identical in two or more sequences.

Now, a process for producing DNA having the promoter activity of thepresent invention, a fragment and a modified version thereof will beexplained below. The DNA having the nucleotide sequence as set forth inSEQ ID NO: 4 was PCR-amplified using a primer AP1(5′-GTAATACGACTCACTATAGGGC-3′) (SEQ ID NO: 5) corresponding to anadapter and the HM1 primer (sequence: 5′-ATC CCC GTC TTC CAT GGG CAC TCTGCA-3′ (SEQ ID NO: 6) corresponding to nucleotide Nos. 47-72 of cDNAclone P3.19 cloned by the above-mentioned panning method, and a humangenomic DNA library as a template, and then, using the PCR-amplifiedproduct as a template, a nested PCR is performed using the AP2 primer(sequence: ACTATAGGGC ACGCGTGGT) (SEQ ID NO: 7) and the HM2 primer(sequence: 5′-ATA GTC ATA CGA AGT AGA TGC CAT CCA G-3′ (SEQ ID NO: 8)corresponding to nucleotides 19-40 of clone P3.19 to subclone into acloning vector pCRII (Invitrogen). By sequencing, DNA having thenucleotide sequence as set forth in SEQ ID NO: 4 was obtained.

A DNA fragment having a promoter can be obtained, for example, in thefollowing manner. A method of digesting a DNA subcloned into the abovecloning vector pCRII with restriction enzymes, ScaI, BamHI, PvuII, PstI,etc., a method using an ultrasonication treatment, a chemical synthesisby the phosphoramidite method, a method of preparation using apolymerase chain reaction method etc. can be used. For example, adesired DNA fragment can be easily prepared by preparing a primer asappropriate from the DNA sequence of SEQ ID NO: 4 and then performing apolymerase chain reaction.

A hybridization method that utilizes the nucleotide sequence of thepromoter of the present invention may be used to obtain a promoter ofthe present invention from a gene derived from other cells. In thiscase, for example, the following method can be used. First, achromosomal DNA obtained from a gene source of other cells is ligatedinto a plasmid or a phage vector and then introduced into a hostaccording to a standard method to construct a library. The library iscultured on a plate, and colonies or plaques grown are transferred to anitrocellulose or a nylon membrane, which is subjected to denaturationto immobilize DNA onto the membrane. The membrane is incubated in asolution containing a probe (as the probe, a DNA fragment as set forthin SEQ ID NO: 4 or a portion thereof) previously labeled with ³²P etc.to form a hybrid between the DNA on the membrane and the probe. Forexample, a DNA-immobilized membrane is subjected to hybridization with aprobe in a solution containing 6×SSC, 1% sodium dodecyl sulfate (SDS),100 μg/ml salmon sperm DNA, 5× Denhardt's at 65° C. for 20 hours. Afterhybridization, non-specific adsorption is washed off, andautoradiography etc. is performed to identify clones that hybridizedwith the probe. The procedure is repeated until a single clone thatformed a hybrid is obtained. Into a clone thus obtained, DNA encoding adesired promoter should be inserted.

The above promoter that has been modified by the deletion, addition,and/or substitution of nucleotides can be prepared by, for example,conventionally known methods such as site-directed mutagenesis, or a PCRmethod.

The gene obtained is sequenced for its nucleotide sequence, for example,in the following manner to confirm the gene obtained is a promoter ofinterest. For the determination of the nucleotide sequence, in the caseof a clone obtained by hybridization, the transformant is cultured in atest tube if it is E. coli, and a plasmid is extracted therefromaccording to a standard method. This is cleaved with a restrictionenzyme to extract an inserted fragment, which is subcloned into M13phage vector etc., and the nucleotide sequence is determined by thedideoxy method or the like.

When the transformant is a phage, essentially similar steps can beemployed to determine the nucleotide sequence. Basic procedures fromculturing to nucleotide sequence determination are carried out asdescribed in, for example, Molecular Cloning: A laboratory Manual,Second edition, T. Maniatis, Chapter One, pp. 90-104, Cold Spring HarborLaboratory, 1989.

Whether the obtained gene is a promoter of interest or not can bedetermined by comparing the determined nucleotide sequence with thepromoter of the present invention and estimating from its homology. Ifthe obtained gene is thought not to contain an entire promoter, asynthetic DNA primer is constructed based on the obtained gene, missingregions are amplified by PCR, and using the obtained gene fragment as aprobe DNA libraries or cDNA libraries are screened so that thenucleotide sequence of the entire conding region of the promoter thathybridizes to the promoter of the present invention can be determined.

The method of expressing useful genes of the present invention ischaracterized in that a DNA fragment obtained by ligating, in a statethat enables expression, a useful gene downstream to the promoter of thepresent invention thus obtained is introduced into an animal cell andthe resulting cell is cultured. In order to ligate, in a state thatenables expression, a useful gene downstream to the DNA fragment of thepromoter of the present invention prepared as above, the DNA ligasemethod or the homopolymer method can be used.

If DNA ligase is used for ligating of the two, they are ligated bydigesting with restriction enzymes and, if they have the samerestriction enzyme site, then both the DNA fragments are mixed in areaction buffer as described in Molecular Cloning: A laboratory Manual,Second edition, T. Maniatis et al. ed., Chapter One, pp. 62, Cold SpringHarbor Laboratory, 1989, and adding DNA ligase thereto or, if the theydo not share the same restriction enzyme site, the ends are blunt-endedwith T4 DNA polymerase (manufactured by Takara), and then treated withDNA ligase as described above.

On the other hand, when the homopolymer method is used, ligating iseffected by attaching a poly G chain to the 3′-end of a vectorlinearized with a restriction enzyme using terminal deoxyribonucleotidyltransferase and dGTP, attaching similarly a poly C chain to the 3′-endof the insert DNA, and then annealing these poly G chain and poly Cchain by, for example, the calcium chloride method for introduction intoE. coli [Proc. Natl. Acad. Sci. U.S.A., 75: 3727 (1978)].

The useful genes of interest that can be used in the present inventionincludes, but not limited to, the interleukin 1-12 gene, the interferonα, β, γ genes, the tumor necrosis factor gene, the colony stimulatingfactor gene, the erythropoietin gene, the transforming growth factor-βgene, the immunoglobulin gene, the tissue plasminogen activator gene,the urokinase gene, the horseradish peroxidase gene, and the like.

There can be mentioned for example, genes of superoxide dismutase, tumornecrosis factor, insulin, calcitonin, somatostatin, secretin, growthhormone, endorphine, viral protein, amylase, lipase, alcoholdehydrogenase, and the like.

The DNA fragment obtained as above in which the DNA fragment of thepresent invention and a useful gene is ligated can be integrated into anappropriate vector to obtain a plasmid for gene expression. Examples ofsuch vectors include pTM [Nucleic Acids Research, 10: 6715 (1982)],cos202 [The EMBO Journal, 6: 355 (1987)], p91203 (B) [Science, 228: 810(1985)], BCMGSNeo [Journal of Experimental Medicine, 172: 969 (1990)],and the like.

The plasmid thus obtained for gene expression can be introduced into asuitable host by the calcium phosphate method [Molecular and CellularBiology, 7: 2745 (1987)], the electroporation method [Proc. Natl. Acad.Sci. U.S.A., 81: 7161 (1984)], the DEAE-dextran method [Methods inNucleic Acids Research, page 283, Column et al., ed., CRC Press, issuedin 1991], the liposome method [BioTechniques, 6: 682 (1989)], and thelike.

Examples of such host cells include COS cells, HeLa cells, CHO cells,BHK-21 cells, and the like. By culturing the resulting transformed cellsin a suitable medium, the useful gene product of interest can beobtained in an efficient manner.

EXAMPLES

The present invention will now be explained in more detail withreference to the following working Examples and Reference Examples.

Reference Example 1 Cloning of cDNA Encoding HM1.24 Antigen Protein

1) Cell Lines

Human multiple myeloma cell lines RPMI8226 and U266 were cultured in aRPMI1640 medium (GIBCO-BRL) supplemented with 10% fetal bovine serum(FBS), and a human multiple myeloma cell line KPMM2 (Japanese UnexaminedPatent Publication (Kokai) No. 7-236475) was cultured in a RPMI1640medium (GIBCO-BRL) supplemented with 20% fetal bovine serum.

2) Construction of cDNA Library

Total RNA was isolated from 1×10⁸ KPMM2 cells by a guanidinethiocyanate/cesium chloride method, and mRNA was purified using the FastTrack mRNA Isolation Kit (Invitrogen). After synthesizing cDNA from 10μg of mRNA using NotI/oligo-dT₁₈ primer (Time Saver cDNA Synthesis Kit;Pharmacia Biotech), an EcoRI adapter was ligated thereto. A cDNA largerthan 0.7 kbp was fractionated using 1.0% low-melting point agarose gel(Sigma), and digested with NotI. It was then inserted into theEcoRI/NotI site of a pCOS1 expression vector or a λExCell vector(Pharmacia Biotech) to prepare a library (library A) for use in directexpression cloning (screening by panning) and a library (library B) foruse in immunoscreening, respectively.

The pCOS1 expression vector was constructed from HEF-PMh-gγl (seeWO92-19759) by deleting the contained gene with EcoRI and SmaIdigestion, and then by ligating the EcoRI-NotI-BamHI Adaptor (TakaraShuzo).

3) Panning

Library A was introduced into COS-7 cells by the electroporation method.Thus, 20 μg of a plasmid DNA (containing 5×10⁵ independent clones) wasmixed with 0.8 ml of cells (1×10⁷ cells/ml in PBS), and the mixture wassubjected to electroporation under a condition of 1.5 kV and 25 μFDcapacity using the Gene Pulser (Bio-Rad). After being allowed to standat room temperature for 10 minutes, the cells were suspended in a DMEM(manufactured by GIBCO-BRL) containing 10% FBS, divided into four 100 mmculture dishes, and cultured at 37° C. for 72 hours.

After culturing, the cells were washed with a phosphate saline buffer(PBS), and were scraped off by adding PBS containing 5 mM EDTA to adjustthe cell suspension to 1 to 2×10⁶ cells/ml in PBS containing 5% FBS and0.02% NaN₃. The cells were then allowed to stand on a panning plate (seebelow) coated with anti-HM1.24 antibody for 2 hours, and the plate wasgently washed three times with 3 ml of PBS containing 5% FBS and 0.02%NaN₃. After washing, plasmid DNA was recovered from the cells bound tothe plate using Hirt's solution (Hitt J., Mol. Biol. 26: 365-369, 1983)(0.6% SDS, 10 mM EDTA). The recovered plasmid DNA was amplified in E.coli and used for the following panning.

A panning plate was prepared as follows. Three milliliters of ananti-HM1.24 antibody solution (10 μg/ml in 50 mM Tris-HCl, pH 9.5) wasadded to a cell culture dish (Falcon) with a diameter of 60 mm and wasincubated at room temperature for 2 hours. After washing three times in0.15 M NaCl, 3 ml of PBS containing 5% FBS, 1 mM EDTA, and 0.02% NaN₃was added to the dish. After blocking by allowing to stand at roomtemperature for 2 hours, the panning plate was stored at −20° C. untiluse.

By repeating panning three times using a plasmid library (library A)containing 5×10⁵ clones as a starting material, a plasmid DNA having anabout 0.9 kbp cDNA as an insert was concentrated. Using a Dye.Terminator Cycle Sequencing Kit (manufactured by Applied Biosystems),the nucleotide sequence was determined using the 373A or 377DNASequencer (Applied Biosystems). The result revealed that clone P3.19comprises 1,012 bp cDNA and has an open reading frame (23-549) encoding180 amino acids (FIGS. 1 and 2) (SEQ ID NO: 1). The amino acid sequencededuced from the cDNA had a structure characteristic to type II membraneproteins and had two N-type sugar chain binding sites.

4) Immunoscreening

Library B was subjected to immunoscreening using anti-HM1.24 antibody.Thus, a phage library containing 1.5×10⁵ independent clones was layeredon agar together with E. coli NM522 (Pharmacia Biotech) and was culturedat 42° C. for 3.5 hours. After culturing, a nitrocellulose filter(Schleicher & Schuell) pretreated with 10 mM IPTG was layered on theplate, and was further cultured at 37° C. for 3 hours. After the filterwas washed with 0.05% (v/v) Tween 20-added TBS (20 mM Tris-HCl, pH 7.4,150 mM NaCl), 1% (w/v) BSA-added TBS was added thereto, and was blockedby incubating at room temperature for 1 hour.

After blocking, an anti-HM1.24 antibody solution (a 10 μg/ml blockingbuffer) was added, incubated at room temperature for 1 hour, and5,000-fold diluted alkaline phosphatase-conjugated anti-mouse Igantiserum (picoBlue Immunoscreening kit; Stratagene) was added, whichwas further incubated at room temperature for 1 hour. Spots that reactedwith the antibody were allowed to develop color with a developingsolution (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl₂) containing0.3 mg/ml nitroblue tetrazolium and 0.15 mg/ml5-bromo-4-chloro-3-indolyl phosphate.

By immunoscreening, five positive clones were isolated, all of whichwere consistent with the partial sequence of P3.19 (FIG. 3). Homologysearch of them revealed that P3.19 is identical with the DNA sequence ofBST-2 (Ishikawa J. et al., Genomics, 26: 527-534, 1995) expressed on thebone marrow or synovial stromal cells. The same molecule was obtainedfrom two types of screening, which strongly suggested that the membraneprotein encoded by P3.19 is the HM1.24 antigen molecule.

E. coli having the plasmid pRS38-pUC19 in which DNA encoding a humanprotein having the same sequence as the above-mentioned human HM1.24antigen protein has been inserted in between the XbaI sites of pUCvector was designated as Escherichia coli DH5α (pRS38-pUC19) and wasinternationally deposited under the provisions of the Budapest Treaty onOct. 5, 1993 with the National Institute of Bioscience and HumanTechnology, Agency of Industrial Science and Technology, of 1-3, Higashi1-chome, Tsukuba-shi, Ibaraki pref., Japan, under an accession No. FERMBP-4434.

5) FACS Analysis

Furthermore, in order to confirm that the protein encoded by p3.19indeed binds to anti-HM1.24 antibody, a CHO transformant cell line inwhich P3.19 was introduced was established. Thus, after the P3.19 clonewas introduced into CHO cells by the electroporation method, it wascultured in the presence of 500 μg/ml G418 (GIBCO-BRL) to obtain a CHOcell line that expresses HM1.24 antigen.

The cultured cells (1×10⁶) were suspended to the FACS buffer (PBS (−)/2%FCS/0.1% NaN₃), HM1.24 antibody was added thereto, which was reacted onice for 30 minutes. After washing in the FACS buffer, it was resuspendedin a GAM-FITC solution (25 μg/ml in the FACS buffer; Becton Dickinson),and was further reacted on ice for 30 minutes. After washing twice withthe FACS buffer, it was resuspended in 600 μl of the FACS buffer formeasurement by the FACScan (Becton Dickinson).

As a negative control, UPC10 was used.

As result of FACS analysis, CHO cells in which P3.19 was introduced wereshown to react strongly with anti-HM1.24 antibody, whereas no bindingwas observed in CHO cells (CHO/NEO) in which the control expressionvector was introduced (FIG. 17). It was confirmed therefore that theprotein encoded by P3.19 binds to anti-HM1.24 antibody.

6) Immunoprecipitation

After washing the cells twice in PBS (−), they were destructed byultrasonication in the cell lysate buffer method (50 mM sodium borate,150 mM NaCl, 0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1 mg/mlphenylmethylsulfonyl fluoride, protease inhibitor cocktail [BoehringerMannheim]) to obtain a soluble fraction. The soluble fraction was addedto anti-HM1.24 antibody-conjugated Sepharose 4B beads. Aftercentrifugation, the precipitate was separated on SDS-PAGE (12% gel),which was transferred onto a PVDF membrane. The PVDF membrane wasreacted with anti-HM1.24 antibody, and then with POD-anti-mouse IgG, anddetected using the ECL kit (Amersham).

Each of myeloma cell lines KPMM2, RPMI8226, and U266 strongly expressedHM1.24 antigen, and the immunoprecipitation of the cell lysates thereofwith anti-HM1.24 antibody allowed the specific detection of protein witha molecular weight of about 29 to 33 kDa (FIG. 5). In a similarexperiment for CHO cell lines (CHO/HM) in which P3.19 was introduced,immunoprecipitants were confirmed in the CHO/HM cells as for the myelomacell lines (FIG. 5, lane 4). Such immunoprecipitants could not beobserved in the control cells (CHO/NEO) in which the expression vectorpCOS1 was only introduced (FIG. 5, lane 5).

P3.19 encodes a protein having an estimated molecular weight of 19.8 kDacomprising 180 amino acids (FIG. 1) and has two N-type sugar chainbinding motifs (FIG. 1). Thus, it suggested that the presence ofsubstances having different molecular weights observed byimmunoprecipitation was due to differences in the modification of N-typesugar chains. In fact, the immunoprecipitants were confirmed to bind toseveral lectins.

Reference Example 2 Preparation of Hybridomas that Produce MouseAnti-HM1.24 Monoclonal Antibody

In accordance with the method of Goto, T. et al., Blood (1994) 84,1992-1930, hybridomas that produce mouse anti-HM1.24 monoclonal antibodywere prepared.

A plasma cell line KPC-32 (1×10⁷) derived from the bone marrow of ahuman patient with multiple myeloma (Goto, T. et al., Jpn. J. Clin.Hematol. (1991) 32, 1400) was injected to the abdominal cavity of aBALB/c mouse (bred by Charles River) twice every six weeks.

Three days prior to sacrificing the animal, 1.5×10⁶ KPC-32 was injectedto the spleen of the mouse in order to further enhance theantibody-producing ability of the mouse (Goto, T. et al., Tokushima J.Exp. Med. (1990) 37, 89). After sacrificing the animal, the spleen wasextracted and the extracted organ was subjected to cell fusion with themyeloma cell SP2/0-according to the method of Groth, de St. &Schreidegger (Cancer Research (1981) 41, 3465).

By Cell ELISA (Posner, M. R. et al., J. Immunol. Methods (1982) 48, 23)using KPC-32, a culture supernatant of a hybridoma was screened forantibody. 5×10⁴ KPC-32 was suspended in 50 ml of PBS and then wasaliquoted to a 96-well plate (U-bottomed, Corning, manufactured byIwaki), which was then air-dried at 37° C. overnight. After blockingwith PBS containing 1% bovine serum albumin (BSA), the culturesupernatant of the hybridoma was added thereto and incubated at 4° C.for 2 hours. Then, peroxidase-labeled anti-mouse IgG goat antibody(manufactured by zymed) was reacted at 4° C. for 1 hour. After washing,o-phenylene diamine solution substrate solution (manufactured bySumitomo Bakelite) was reacted at room temperature for 30 minutes.

Reaction was stopped by adding 2 N sulfuric acid and the absorbance wasmeasured at 492 nm using the ELISA reader (manufactured by Bio-Rad). Inorder to remove the hybridomas that produce antibodies against humanimmunoglobulin, a culture supernatant of positive hybridomas hadpreviously been adsorbed to human serum and the reactivity to other celllines was screened by ELISA. Positive hybridomas were selected, andtheir reactivity to various cells was investigated by flow cytometry.The last selected hybridoma clone was cloned twice and injected to theabdominal cavity of a pristane-treated BALB/c mouse. The ascites wasobtained from the mouse.

Monoclonal antibody was purified from the ascites of the mouse byammonium sulfate precipitation and a Protein A affinity chromatographykit (Ampure Pa., manufactured by Amersham). The purified antibody waslabeled with FITC using the Quick Tag FITC biding kit (manufactured byBoehringer Mannheim).

As a result, monoclonal antibodies produced by 30 hybridoma clonesreacted with KPC-32 and RPMI 8226. After cloning, the reactivity of theculture supernatants 2.0 of these hybridomas with other cell lines orperipheral blood mononuclear cells was investigated.

Of them, 3 clones were monoclonal antibodies that specifically reactedwith the plasma cell. From the 3 clones, a hybridoma clone that was mostuseful for flow cytometry analysis and had a CDC activity to RPMI 8226was selected and designated as HM1.24. The subclass of the monoclonalantibody produced by this hybridoma was determined by an ELISA using asubclass-specific anti-mouse rabbit antibody (manufactured by Zymed).Anti-HM1.24 antibody had a subclass of IgG2a κ. The hybridoma HM1.24that produces anti-HM1.24 antibody was internationally deposited underthe provisions of the Budapest Treaty as FERM BP-5233 on Sep. 14, 1995with the National Institute of Bioscience and Human Technology, Agencyof Industrial Science and Technology, of 1-3, Higashi 1-chome,Tsukuba-shi, Ibaraki pref., Japan.

Example 1 Cloning of the Promoter Region of HM1.24 Antigen Gene

Since HM1.24 antigen was strongly expressed in all the myeloma cellsanalyzed so far, it is very likely that the expression of HM1.24 antigenis deeply involved in physiological characteristics of multiple myeloma.Thus, the elucidation of the mechanism of HM1.24 antigen expression isan important challenge, and the inventors have clarified the genestructure of the promoter region.

The promoter region of the HM1.24 antigen gene was isolated using thePromoterFinder DNA Walking kit (Clontech). From the nucleotide sequenceof the 5′-end of clone P3.19 isolated by Panning, two PCR primers weredesigned: HM1 (5′-ATC CCC GTC TTC CAT GGG CAC TCT GCA-3′) (SEQ ID NO: 6)and HM2 (5′-ATA GTC ATA CGA AGT AGA TGC CAT CCA G-3′) (SEQ ID NO: 8).The first PCR was performed using primer AP1 (attached to the kit)corresponding to the adapter and the HM1 primer according to theinstruction manual attached to the kit, and then the PCR product wassubjected to a nested PCR using the AP2 primer (attached to the kit) andthe HM2 primer. After the final PCR product was purified, it wassubcloned into the pCRII cloning vector (Invitrogen).

The promoter region gene was simply isolated by the. PCR method. Thus,PCR products of about 2.0 kb, 0.7 kb, and 0.3 kb were specificallyamplified from the EcoRV, PvuII, and DraI libraries (Promoter FinderKit; Clontech), respectively. They were demonstrated to be derived fromthe same genomic DNA based on the cleavage patterns with restrictionenzymes (FIG. 6). As a result of sequencing the nucleotide sequences,gene sequence of cDNA from 5′-end to 1959 bp upstream was determined(FIGS. 7 and 8) (SEQ ID NO: 4). By a binding motif search of knowntranscription factors, the presence of transcription controllingelements of AP-2, Sp1, NF-IL6, NF-κB, STAT3 or ISGF3, and the like wasobserved, suggesting the possibility that the expression is controlledby the stimulation by inflammatory cytokeines such as IL-6 or IFN-α.

IL-6 is known to serve as a growth factor of myeloma cells, andtherefore it was strongly suggested that NF-IL6 and STAT3 that aretranscription factors acting downstream of IL-6 are involved in theexpression control of HM1.24 antigen in myeloma cells (FIGS. 7 and 8).The transcription initiation point was estimated nucleotide on thenucleotide sequence of PCR products amplified using the CapSwitcholigonucleotide (CapFnder Kit; Clonetech), and at 27 positions upstreamthereof a TATA box-like sequence (TAATAAA) was observed (FIGS. 7 and 8).

Example 2 Cloning of Genomic DNA for HM1.24 Antigen

Genomic DNA for HM1.24 antigen was amplified from human genomic DNA(Clontech) prepared from a human genomic DNA library (Promoter FinderDNA walking kit; Clontech) or peripheral blood using each PCR primershown in FIG. 9. After purification, PCR products were each subclonedinto the pCRII vector and the nucleotide sequence was determined.

Genomic DNA encoding HM1.24 antigen was divided into four fragments,which were amplified from human genomic DNA prepared from a humangenomic DNA library (Promoter Finder kit; Clontech) or human peripheralblood (FIG. 9). After confirming their nucleotide sequences, they werecompared to the nucleotide sequence of HM1.24 antigen cDNA with a resultthat the HM1.24 antigen gene is composed of four exons and three intronsof 850 bp, 183 bp, and 307 bp (FIG. 10).

However, from the human genomic DNA library prepared from the humanplacenta tissue, a gene consisting of 3 exons lacking intron 3 was onlyamplified, suggesting the presence of a genomic gene having a differentexon/intron structure. In any structure, two N-type sugar chain bindingsites and three cysteine residues present in the extracellular region ofHM1.24 antigen were all present in exon I (FIGS. 11 and 12) (SEQ ID NO:2).

Example 3 Confirmation of HM1.24 Antigen Splicing Variants

In order to confirm the presence of splicing variants of HM1.24 antigen,HM1.24 antigen cDNA was amplified by the PCR method using as a templatecDNA prepared from a human myeloma cell line KPMM2 according to themethod described above. The sense primer BST2-N (SEQ ID NO: 17; ATG GCATCT ACT TCG TAT GAC) used in the PCR corresponds to bases 10 to 30 ofP3.19 (SEQ ID NO: 1), isolated herein, and the antisense primer S3 (SEQID NO: 18; AAC CGT GTT GCC CCA TGA) corresponds to bases 641-658 ofP3.19.

PCR-amplified products were subcloned into a cloning vector pCRII(Invtrogen) and from the resulting independent clones, plasmid DNA wasrecovered, with a result that two inserts having different sizes ofabout 650 bp and about 550 bp were observed. After the determination ofeach nucleotide sequence, it was found that the insert of about 650 bphad the sequence identical with that of P3.19 whereas the insert ofabout 550 bp had a deletion corresponding to bases 294 to 422 of P3.19(SEQ ID NO: 19). The region in which the deletion was observedcorresponds to exons 2 and 3 of human HM1.24 antigen genomic DNA,indicating the presence of variants due to different splicing.

Example 4 Analysis of Polymorphism of HM1.24 Gene

In connection with polymorphism found in the HM1.24 gene, itsrelationship to multiple myeloma was investigated. The peripheral bloodsamples of normal healthy humans were supplied as the buffy coat ofdonated blood samples from Japan Red Cross Tokushima Blood Center. Forpatients with myeloma, the peripheral blood or bone marrow fluid wascollected from patients in Tokushima University First Internal MedicineHospital or affiliated hospitals. Blood samples were subjected to theFicoll-Conrey density centrifugation to separate mononuclear cells.Myeloma cell lines were cultured in a RPMI1640 medium (GIBCO-BRL,Rockville, Md., U.S.A.) containing 10% fetal bovine serum at 37° C. in a5% CO₂ incubator. The peripheral blood mononuclear cells or the myelomacell lines were treated with the DNAzol reagent (GIBCO-BRL) according tothe protocol to extract genomic DNA from the cells.

The nucleotide sequence was determined by the PCR-direct sequencingmethod. 5′-promoter region was amplified by PCR (30 cycles of 94° C. for1 minute, 55° C. for 1 minute, and 72° C. for 1 minute) with ampliTaqDNA polymerase (Perkin Elmer, Chiba) using primer 6S(TCCATAGTCCCCTCGGTGG) (SEQ ID NO: 22) and BST2B(ATAGTCATACGAAGTAGATGCCATCCAG) (SEQ ID NO: 23). The HM coding region wasamplified by PCR with LA Taq DNA Polymerase (Takara Shuzo, Otsu) usingprimer HMP2K (AAAGGTACCAGCTGTCTTTCTGTCTGTC) (SEQ ID NO: 24) and BST2-R4(GTGCTCTCCCCGCTAACC) (SEQ ID NO: 25). With the reaction mixture as atemplate, PCR was further performed with Ex Taq DNA polymerase (TakaraShuzo) using primer 8S (GGACGTTTCCTATGCTAA) (SEQ ID NO:26) and BST2-R1(AAAGCGGCCGCTCATCACTGCAGCAGAGCGCTGAG) (SEQ ID NO: 27)).

The reaction mixture was purified by the QIA Quick PCR Purification Kit(QIAGEN, Tokyo), and reacted with the resulting PCR fragment as atemplate, using, as a primer, 6S or BST2B for the 5′-promoter region,and 8S, HMINTIF (AGGGGAACTCACCAGACC) (SEQ ID NO: 28), HMEX2F(ATGGCCCTAATGGCTTCC) (SEQ ID NO: 29), HMEX3F (CATTAAACCATAAGCTTCAGG)(SEQ ID NO: 30), HMEX2R (CCCTCAAGCTCCTCCACT) (SEQ ID NO: 31), or BST2-R1for the HM coding region by the BigDye Terminator Cycle Sequencing Kit(Perkin Elmer). The nucleotide sequence was determined using the ABI3777DNA Sequencer (Perkin Elmer). The frequency of 8 base pair deletion inthe vicinity of 20 base pairs upstream to the initiation codon of theHM1.24 gene was detected by PCR. Thus, PCR (30 cycles of 94° C. for 1minute, 55° C. for 1 minute, and 72° C. for 1 minute) was performed withampliTaq DNA polymerase (Perkin Elmer, Chiba) using primers 85 andBST-R3 (GACGGATCCTAAAGCTTACAGCGCTTATC) (SEQ ID NO: 32). The reactionmixture was electrophoresed on a 4% agarose gel and detected byethidiumbromide staining.

Polymorphism of the 5′-Promoter Region of the HM1.24 Gene

For samples from normal healthy humans and patients, the nucleotidesequence of the 5′-promoter region of the HM1.24 gene was determined.The result is shown in FIGS. 14-18 (SEQ ID NO: 33). There were samplesfor which nucleotide substitution in the underlined 187, 262, and 323 inFIG. 14, and deletions near 360 in FIG. 14 and near 555 in FIG. 15 wereobserved, and there was a sample for which the region of 366 to 558could not be decoded. When the sequence described in FIGS. 14 to 18 (SEQID NO: 33) was termed as type A, and the mutation type having the abovenucleotide substitution/deletion as type B, the sample for which theregion of 366 to 558 could not be decoded is thought to be aheterozygote (AB) of A and B, and the sample that could be decoded astype A or B is thought to be a homozygote (AA) of A or a homozygote (BB)of B. In addition to the above polymorphism, it was clarified that 19 bpwas inserted at tandem at the double-underlined region in a myeloma cellline HS-sultan (type M).

Polymorphism of the HM1.24 Gene

For the genomic gene region of cell lines of type AA (U266, HS-sultan)and two samples of type BB from normal healthy humans, the nucleotidesequence was determined. As a result, it was found that 3 bases of cwere missing near 2315 of intron 3 in the sequence of type B, whereas nomutation was observed in the coding region.

Gene Frequency of Type A and Type B

Polymorphism and disease sensitivity were investigated. When 8-basedeletion was detected by PCR near 20 base pairs upstream to theinitiation codon of the HM1.24 gene, there was no difference infrequency of polymorphism between 94 cases of normal healthy humans and46 cases of patients with myeloma (Table 1). In both the normal healthyhumans and the patients with myeloma, type A gene was dominant as thegene distribution was about A:B=2:1. For cell lines, there was a bias totype A with 9 cases out of 11 being type AA.

No relationships were observed between the polymorphism of the HM1.24promoter region and sensitivity to myeloma diseases.

TABLE 1 Frequency of polymorphism AA AB BB Total Normal healthy humans43 37 14 94 (%) 45.7 39.4 14.9 Myeloma patients 21 21 4 46 (%) 45.7 45.78.7 Myeloma cell lines 9 2 0 (%) 81.8 18.2 0

Industrial Applicability

According to the present invention, there was obtained the genomic geneof HM1.24 antigen that is highly expressed in all myeloma cells. Thegenomic gene that encodes HM1.24 antigen is useful for analysis ofHM1.24 antigen. Since HM1.24 antigen is strongly expressed, the promoterregion is thought to have a strong promoter activity, and accordingly isuseful for the expression of useful genes.

Reference to the microorganisms deposited under the Patent CooperationTreaty, Rule 13-2, and the name of the Depository organ

Depository Organ

-   Name: the National Institute of Bioscience and Human Technology,    Agency of Industrial Science and Technology Address: 1-3, Higashi    1-chome, Tsukuba city, Ibaraki    -   pref., Japan        Organism (1)

Name: Escherichia coli DH5α (pRS38-pUC19)

Accession number: FERM BP-4434

Date deposited: Oct. 5, 1993

Organism (2)

Name: Mouse-mouse hybridoma HM1.24

Accession number: FERM BP-5233

Date deposited: Sep. 14, 1995

1. A recombinant promoter sequence DNA having the nucleotide sequence ofthe 5′-non-coding region as set forth in SEQ ID NO:
 4. 2. A recombinantDNA wherein a nucleic acid is operably linked to the DNA having apromoter activity according to claim
 1. 3. The recombinant DNA accordingto claim 2 wherein said nucleic acid encodes a useful protein.
 4. Avector comprising the recombinant DNA according to claim
 2. 5. Thevector according to claim 4 wherein the vector is a plasmid vector or avirus vector.
 6. An isolated animal cell into which the recombinant DNAaccording to claim 2 has been introduced.
 7. An isolated animal celltransformed with the vector according to claim
 4. 8. A method ofexpressing a gene which method comprises culturing an isolated animalcell into which the recombinant DNA according to claim 3 has beenintroduced.
 9. A method of producing a protein which method comprisesculturing an isolated animal cell transformed with an expression vectorcomprising a nucleic acid encoding a useful protein operably linked tothe DNA according to claim
 1. 10. A vector comprising the recombinantDNA according to claim
 3. 11. The vector according to claim 10 whereinthe vector is a plasmid vector or a virus vector.